Genetically engineered yeast

ABSTRACT

A genetically modified  Saccharomyces cerevisiae  including an active fermentation pathway producing 3-HP expresses an exogenous gene expressing the aminotransferase YhxA from  Bacillus cereus  AH1272 catalysing a transamination reaction between beta-alanine and pyruvate to produce malonate semialdehyde. The yeast may also express a 3-hydroxyisobutyrate dehydrogenase (HIBADH) and a 3-hydroxypropanoate dehydrogenase (3-HPDH) and aspartate 1-decarboxylase. Additionally the yeast may express pyruvate carboxylase and aspartate aminotransferase.

The present invention relates to genetically engineered yeasts and theiruse in methods for production of 3-hydroxypropionic acid (3HP).

3HP is a platform chemical, which can be converted to acrylic acid,1,3-propandiol, malonic acid, and other valuable products. Acrylicacid-derived products include superabsorbent polymers used in babydiapers and incontinence products, various plastics, coatings,adhesives, elastomers, and paints. Currently acrylic acid is derivedfrom propylene, a by-product of ethylene and gasoline production.Establishment of 3HP production from glucose or other renewable carbonsource would provide a biosustainable alternative to acrylic acidproduction from fossil resources. Several methods for production of 3HPfrom glucose have been described. The specific teachings howeverprimarily use the bacterium Escherichia coli as the host. The presentinvention uses yeast as the host for 3HP production. This allowsexecuting the process at low pH and thus makes it overall moreeconomical.

US2010/0136638 describes, in general terms, production of 3-HP inmicro-organisms including yeast by biocatalysis from beta-alanine. It issaid that beta-alanine can be synthesized in cells from alpha-alanine byan enzyme having alanine 2,3-aminomutase activity, and sequences aregiven for relevant enzymes.

Also disclosed are methods of producing 3-HP from beta-alanine usingbeta-alanine/pyruvate aminotransferase (BAPAT) sequences. Transformedcells having BAPAT activity, which allows the cell to convertbeta-alanine to 3-HP through a malonate semialdehyde intermediate, aredisclosed.

Although the possibility of conducting such work in yeast is mentioned,there is no practical demonstration of this. We have found that enzymesin this pathway that are effective in E. coli are not effective inSaccharomyces cerevisiae. In particular, according to US2010/0136638enzymes having BAPAT activity can be obtained from Pseudomonas putida orPseudomonas aeruginosa. However, we have found that genes encoding theseenzymes are not effective in S. cerevisiae.

Malonate semialdehyde (or malonic semialdehyde or 3-oxopropanoic acid)is a key intermediate in one pathway leading to 3HP, but many differentroutes to its production are possible.

US2012135481 describes a 3HP producing pathway in yeast including genesencoding gabT, 3-HPDH and HIBADH and others. However, other and better3HP producing yeasts are needed.

We have now found that 3HP production from beta-alanine was obtained inyeast S. cerevisiae when an uncharacterized aminotransferase yhxA fromBacillus cereus AH1272 was heterologously expressed. The amino acidsequence of the said yhxA encoded aminotransferase is set out in SEQ IDNO1 and the DNA sequence is set out in SEQ ID NO2. SEQ ID NO2 iscodon-optimized for S. cerevisiae.

It is our belief that the said aminotransferase YhxA from Bacilluscereus AH1272 catalyzes a transamination reaction between beta-alanineand pyruvate leading to L-alanine and malonic semialdehyde, in whichcase the enzyme would be beta-alanine-pyruvate aminotransferase E.C.2.6.1.18 (BAPAT) rather than a gabT (E.C. 2.6.1.19).

US2012/0135481 discloses genetically modified yeast cells comprising anactive 3-HP fermentation pathway including the BAAT gene (beta alanineamino transferase—EC 2.6.1.19) which catalyzes the conversion of[beta]-alanine to malonate semialdehyde. BAAT here is thereforesynonymous with naturally occurring or genetically modified gabT.However, successful production of 3-HP by this method is not shown.

WO2005/118719 discloses, but does not demonstrate the effectiveness of,methods of producing 3-HP from beta-alanine using beta-alanine/pyruvateaminotransferase (BAPAT) sequences from any organism in a yeast cell.Identified sources for BAPAT here include Pseudomonas, Arabidopsis, ratand Xenopus. As mentioned above, we have established that a BAPAT genesfrom Pseudomonas is not effective in S. cerevisiae.

The Uniprot entry for yhxA provides a sequence but does not identify theenzyme as being a BAPAT.

Accordingly, the present invention now provides a genetically modifiedyeast cell comprising an active fermentation pathway producing 3-HP,wherein the cell comprises and expresses an exogenous gene coding forthe production of an enzyme having at least 80% identity with SEQ ID NO:1 and catalysing a transamination reaction between beta-alanine andpyruvate to produce malonate semialdehyde.

Preferably, said yeast also expresses 3-hydroxyisobutyrate dehydrogenase(HIBADH), suitably from Pseudomonas aeruginosa, P. putida, Bacilluscereus, or Candida albicans and/or 3-hydroxypropanoate dehydrogenase(3-HPDH), optionally from Metallosphaera sedula, Sulfolobus tokadaii orE. coli.

To enable the synthesis of 3-hydroxypropionic acid directly from glucoseis it preferred in addition to reconstructing pathway from beta-alanineto 3-hydroxypropionic acid to express heterologous aspartate1-decarboxylase, preferably from insect, preferably red flour beetle(Tribolium castaneum). To further increase the flux towards3-hydroxypropinic acid it is preferred to overexpress pyruvatecarboxylase and or PEP carboxylase and aspartate aminotransferase.Additionally deletion of pyruvate decarboxylase activity (PDC1, PDC5,PDC6) or alcohol dehydrogenase (ADH) activity would allow anaerobicfermentation without formation of ethanol as a by-product.

Strains according to the invention can be evolved using adaptivelaboratory evolution methods to improve glucose tolerance, removeacetate dependence and increase 3HP production.

The yeast is preferably S. cerevisiae but may be Saccharomyces kluyveri,Yarrowia lipolytica, Schizosaccharomyces pombe, Debaryomyces hansenii,Cyberlindnera jadinii, Rhodotula minuta, Rhodotula glutinis, Torulasporadelbrueckii, Pichia stipitis, Pichia pastoris, Kluyveromyces lactis,Kluyveromyces marxianus, or other yeast.

Yeast strains suitable for modification according to the invention canbe selected for their tolerance to growth in the presence of 3HP.

The amino acid sequence of the native yhxA expression product of B.cereus AH1272 and the DNA sequence coding for it can be modified for usein this invention in various ways. First, the DNA sequence can be codonoptimised for expression in the appropriate yeast. Secondly, the aminoacid sequence may be modified by deletion, addition, or substitution ofamino acids whilst not interfering with, or indeed whilst increasing,the enzyme activity. Such a modified enzyme may have at least 80%, morepreferably at least 85%, or 90% or 95% homology with the native aminoacid sequence.

The invention includes a method for the production of 3HP comprisingculturing a yeast cell of the invention, and optionally recovering 3HPfrom the culture. The culture may be conducted in a culture mediumincluding beta-alanine or a source thereof other than said yeast. Saidsource may be another micro-organism. However, the yeast of theinvention may be engineered to produce beta-alanine, e.g. fromL-aspartate, suitably by incorporating exogenous genes producingaspartate-1-decarboxylase (EC 4.1.1.11) or glutamate decarboxylase (EC4.1.1.15) or from L-alanine by 2,3-alanine aminomutase. Due to its rolein the synthesis of pantothenate, aspartate 1-decarboxylase is alsoknown as PanD. A gene for this enzyme is not present in the genome ofwild-type S. cerevisiae.

We have found that superior results are obtained using certain exogenousPanD genes encoding aspartate-1-decarboxylase compared to others. Inparticular, we have found that PanD genes from insects, especially flourbeetles, more especially red flour beetle (Tribolium castaneum),provides better production titres and better yields of 3-HP compared tobacterial PanD genes.

Preferably, the production of 3HP by said yeast is such that at least100 mg of 3HP per litre of culture medium is produced or is recoveredfrom said culture medium, more preferably at least 200, or 300, or 400or 500 or 1000 or 2000 or 14000 mg/l.

The invention will be further described and illustrated in the followingnon-limiting examples, in which reference will be made to the followingTables.

TABLE 1 Primers Primer name Primer sequence, 5′ → 3′ pE1_fwAGTGCAGGU GGTACCAAAACAATG SEQ ID NO 26 pE1_rv CGTGCGAU GTCGACTCASEQ ID NO 27 EcRutE_U1_fw AGTGCAGGU AAAACAATGAACGAAGCCGTTAG SEQ ID NO 28EcRutE_U1_rv CGTGCGAU TTACAACAGCCCGCAG SEQ ID NO 29 EcYdfG_U1_fwAGTGCAGGU AAAACAATGATCGTTTTAGTAACTGG SEQ ID NO 30 EcYdfG_U1_rvCGTGCGAU TTACTGACGGTGGACATTC SEQ ID NO 31 scGabT_U1_fwAGTGCAGGU AAAACAATGTCTATTTGTGAACAATA SEQ ID NO 32 CTAC ScGabT_U1_rvCGTGCGAU TCATAATTCATTAACTGATTTGG SEQ ID NO 33 GeneArt_1U_fwAGTGCAGGU GCATGGTACCAAAACAATG SEQ ID NO 34 GeneArt_1U_rvCGTGCGAU ATGAGGCCCAGGTCGAC SEQ ID NO 35 PTEF1_fwACCTGCACU TTGTAATTAAAACTTAG SEQ ID NO 36 PTEF1_rvCACGCGAU GCACACACCATAGCTTC SEQ ID NO 37 ydfG_KpnI_AAAA GGTACC ATGATCGTTTTAGTAACTGG SEQ ID NO 38 express_fw ydfG_PacI_AAAA TTAATT AATTACTGACGGTGGACATTC SEQ ID NO 39 express_rv EcPAND_U1_fwAGTGCAGGU AAAACAATGATCAGAACCATG SEQ ID NO 40 EcPAND_U1_rvCGTGCGAU TCAAGCAACTTGAACTGG SEQ ID NO 41 CgPAND_U1_fwAGTGCAGGU AAAACAATGTTGAGAACC SEQ ID NO 42 CgPAND_U1_rvCGTGCGAU TCAAATGGATCTAGAAGTC SEQ ID NO 43 RnGAD1_U1_fwAGTGCAGGU AAAACAATGGCTTCTTCTACTC SEQ ID NO 44 RnGAD1_U1_rvCGTGCGAU TCACAAATCTTGACCCAATC SEQ ID NO 45 ScGAD1_U1_fwAGTGCAGGU AAAACAATGTTACACAGGCACGGTTC SEQ ID NO 46 ScGAD1_U1_rvCGTGCGAU TCAACATGTTCCTCTATAGTTTCTC SEQ ID NO 47 EcGAD1_U1_fwAGTGCAGGU AAAACAATGGACCAGAAGCTGTTAAC SEQ ID NO 48 EcGAD1_U1_rvCGTGCGAU TCAGGTGTGTTTAAAGCTG SEQ ID NO 49 pE2_fwATCTGTCAU GGTACCAAAACAATG SEQ ID NO 60 pE2_rv CACGCGAU GTCGACTCASEQ ID NO 61 EcYdfg_U2_fw ATCTGTCAU AAAACAATGATCGTTTTAGTAACTGGAGSEQ ID NO 62 EcYdfg_U2_rv CACGCGAU TTACTGACGGTGGACATTC SEQ ID NO 63PTEF1_fw ACCTGCACU TTGTAATTAAAACTTAG SEQ ID NO 64 PPGK1_rvATGACAGAU TTGTTTTATATTTGTTG SEQ ID NO 65 TcPAND_U1_fwAGTGCAGGU AAAACAATGCCAGCTACTGGTG SEQ ID 70 TcPAND_U1_rvCGTGCGAU TCACAAATCGGAACCCAATC SEQ ID 71 ScPYC1_U1_fwAGTGCAGGU AAAACA ATGTCGCAAAGAAAATTCG SEQ ID 72 ScPYC1_U1_rvCGTGCGAU TCATGCCTTAGTTTCAACAG SEQ ID 73 ScPYC2_U2_fwATCTGTCAU AAAACA ATGAGCAGTAGCAAGAAATTG SEQ ID 74 ScPYC2_U2_rvCACGCGAUTTACTTTTTTTGGGATGGG SEQ ID 75 ScAAT2_U1_fwAGTGCAGGU AAAACA ATGTCTGCCACTCTGTTCA SEQ ID 76 ScAAT2_U1_rvCGTGCGAU TTACAATTTAGCTTCAATAGTATAG SEQ ID 77

TABLE 2 Intermediate plasmids Plasmid name Parent plasmid Synthetic genesequence cloned pE1-PpBAPAT pE1 SEQ ID NO4 pE1-PaHIBADH pE1 SEQ ID NO6pE1-CaHIBADH pE1 SEQ ID NO8 pE1-PpHIBADH pE1 SEQ ID NO10 pE1-BcHIBADHpE1 SEQ ID NO12 pE1-MsHPDH pE1 SEQ ID NO14 pE1-StMSR pE1 SEQ ID NO16pE1-CaGabT pE1 SEQ ID NO18 pE2-MsHPDH pE2 SEQ ID NO14

TABLE 3 Primers and templates used to generate gene fragments for USERcloning by PCR Fragment name Gene Fw_primer Rv_primer Template DNAPaHIBADH<- 3-hydroxyisobutyrate pE1_fw pE1_rv pE1-PaHIBADH dehydrogenasefrom Pseudomonas aeruginosa CaHIBADH<- 3-hydroxyisobutyrate pE1_fwpE1_rv pE1-CaHIBADH dehydrogenase from Candida albicans BcHIBADH<-3-hydroxyisobutyrate pE1_fw pE1_rv pE1-BcHIBADH dehydrogenase fromBacillus cereus PpHIBADH<- 3-hydroxyisobutyrate pE1_fw pE1_rvpE1-PpHIBADH dehydrogenase from Pseudomonas putida MsHPDH<-3-hydroxypropanoate pE1_fw pE1_rv pE1-MsHPDH dehydrogenase fromMetallosphaera sedula StMSR<- 3-hydroxypropanoate pE1_fw pE1_rvpE1-StMSR dehydrogenase from Sulfolobus tokadaii EcRutE<-3-hydroxypropanoate EcRutE_U1_fw EcRutE_U1_rv gDNA of E. dehydrogenasefrom coli Escherichia coli SEQ ID NO20 EcYdfG<- 3-hydroxypropanoateEcYdfG_U1_fw EcYdfG_U1_rv gDNA of E. dehydrogenase from coli Escherichiacoli SEQ ID NO22 PpBAPAT<- Beta-alanine-pyruvate pE1_fw pE1_rvpE1-PpBAPAT aminotransferase from Pseudomonas putida KT2440 BcBAPAT<-Uncharacterized GeneArt_1U_fw GeneArt_1U_rv GeneArt aminotransferaseyhxA plasmid with from Bacillus cereus synthetic AH1272 gene sequenceSEQ ID NO2. ScGabT<- Gamma-aminobutyrate ScGabT_U1_fw ScGabT_U1_rv gDNAof S. transaminase ugal from cerevisiae S. cerevisiae CEN.PK113-7D SEQID NO24 CaGabT<- Gamma-aminobutyrate pE1_fw pE1_rv pE1-CaGabTtransaminase from Clostridium acetobutylicum ATCC 824 (as control)ScPTEF1<- Promoter of tef1 gene PTEF1_fw PTEF1_rv gDNA of S. from S.cerevisiae cerevisiae CEN.PK113-7D SEQ ID NO25 EcPanD<- Aspartate 1-EcPAND_U1_fw EcPAND_U1_rv gBLOCK from decarboxylase panD Integrated fromE. coli DNA Technologies SEQ ID NO50 CgPanD<- Aspartate 1- CgPAND_U1_fwCgPAND_U1_rv gBLOCK from decarboxylase panD Integrated from C.glutamicum DNA Technologies SEQ ID NO51 ScGAD1<- Glutamate ScGAD1_U1_fwScGAD1_U1_rv gDNA of S. decarboxylase gad1 cerevisiae from S. cerevisiaeCEN.PK113-7D SEQ ID NO52 EcGAD1<- Glutamate EcGAD1_U1_fw EcGAD1_U1_rvgDNA of E. decarboxylase gad1 coli from E. coli SEQ ID NO53 RnGAD1<-Glutamate RnGAD1_U1_fw RnGAD1_U1_rv GeneArt decarboxylase gad1 plasmidwith from R. norvegicus synthetic gene sequence SEQ ID NO54 MsHPDH->3-hydroxypropanoate pE2_fw pE2_rv pE2-MsHPDH dehydrogenase fromMetallosphaera sedula EcYdfG-> 3-hydroxypropanoate EcYdfG_U2_fwEcYdfG_U2_rv gDNA of E. dehydrogenase from coli Escherichia coli SEQ IDNO22 <-ScPTEF1-ScPPGK1-> Fused promoters of PTEF1_fw PPGK1_rv plasmidpSP- tefl and pgkl genes GM1 SEQ ID from S. cerevisiae NO 66 TcPanD<-Aspartate 1- TcPAND_U1_fw TcPAND_U1_rv GeneArt decarboxylase from T.plasmid with castaneum synthetic gene sequence SEQ ID 69 ScPYC1<-Pyruvate carboxylase ScPYC1_U1_fw ScPYCl_U1_rv gDNA of S. PYC1 from S.cerevisiae cerevisiae CEN.PK113-7D SEQ ID 78 ScPYC2-> Pyruvatecarboxylase ScPYC2_U2_fw ScPYC2_U2_rv gDNA of S. PYC2 from S. cerevisiaecerevisiae CEN.PK113-7D SEQ ID 79 ScAAT2<- Aspartate ScAAT2_U1_fwScAAT2_U1_rv gDNA of S. aminotransferase AAT2 cerevisiae from S.cerevisiae CEN.PK113-7D SEQ ID 80

TABLE 4 Expression plasmids Cloned Selection fragment Plasmid nameParent plasmid marker (-s) Promoter Terminator pPaHIBADH pESC-HIS-USERSpHIS5 PaHIBADH<- ScPTEF1<- ScTADH1 pCaHIBADH pESC-HIS-USER SpHIS5CaHIBADH<- ScPTEF1<- ScTADH1 pBcHIBADH pESC-HIS-USER SpHIS5 BcHIBADH<-ScPTEF1<- ScTADH1 pPpHIBADH pESC-HIS-USER SpHIS5 PpHIBADH<- ScPTEF1<-ScTADH1 pMsHPDH pESC-HIS-USER SpHIS5 MsHPDH<- ScPTEF1<- ScTADH1 pStMSRpESC-HIS-USER SpHIS5 StMSR<- ScPTEF1<- ScTADH1 pEcRutE pESC-HIS-USERSpHIS5 EcRutE<- ScPTEF1<- ScTADH1 pEcYdfG pESC-HIS-USER SpHIS5 EcYdfG<-ScPTEF1<- ScTADH1 pPpBAPAT pESC-URA-USER KlURA3 PpBAPAT<- ScPTEF1<-ScTADH1 pBcBAPAT pESC-URA-USER KlURA3 BcBAPAT<- ScPTEF1<- ScTADH1pScGabT pESC-LEU-USER KlURA2 ScGabT<- ScPTEF1<- ScTADH1 pCaGabTpESC-LEU-USER KlURA2 CaGabT<- ScPTEF1<- ScTADH1 pESC-URA-BcBAPAT-pEEG-URA-USER KlURA3 BcBAPAT<-, <-ScPTEF1- ScTADH1, MsHDPH MsHPDH->ScPPGK1-> ScTCYC1 pESC-URA-BcBAPAT- pESC-URA-USER KlURA3 BcBAPAT<-,<-ScPTEF1- ScTADH1, EcYdfG EcYdfG-> ScPPGK1-> ScTCYC1 pESC-HIS-EcPanDpESC-HIS-USER SpHIS5 EcPanD<- ScPTEF1<- ScTADH1 pESC-HIS-CgPanDpESC-HIS-USER SpHIS5 CgPanD<- ScPTEF1<- ScTADH1 pESC-HIS-TcPanDpESC-HIS-USER SpHIS5 TcPanD<- ScPTEF1<- ScTADH1 pESC-HIS-ScGAD1pESC-HIS-USER SpHIS5 ScGAD1<- ScPTEF1<- ScTADH1 pESC-HIS-EcGAD1pESC-HIS-USER SpHIS5 EcGAD1<- ScPTEF1<- ScTADH1 pESC-HIS-RnGAD1pESC-HIS-USER SpHIS5 RnGAD1<- ScPTEF1<- ScTADH1 pXI-1-LoxP-KlLEU2-pXI-1-LoxP- KlLEU2 ScPYC1<- <-ScPTEF1- ScTADH1, PYC1<-PTEF1-PPGK1->KlLEU2 (SEQ ID ScPYC2-> ScPPGK1-> ScTCYC1 PYC2 NO 87) pX-2-LoxP-KlURA3-pX-2-LoxP- BcBAPAT<-, <-ScPTEF1- ScTADH1, BcBAPAT<-PTEF1- KlURA3 (SEQ IDKlURA3 EcYdfG-> ScPPGK1-> ScTCYC1 PPGK1->EcYdfG NO 86) pTY-BcBAPAT<-pTY* KlURA3- BcBAPAT<-, <-ScPTEF1- ScTADH1, PTEF1-PPGK1-> taggedEcYdfG-> ScPPGK1-> ScTCYC1 EcYdfG pTY-TcPanD<-PTEF1 pTY KlURA3- TcPanD<-ScPTEF1<- ScTADH1 tagged pX-4-LoxP-SpHIS5- pX-4-LoxP- SpHIS5 TcPanD<-TcPanD<-PTEF1 SpHIS5 (SEQ ID ScPTEF1<- ScTADH1 NO 89) pX-4-LoxP-SpHIS5-pX-4-LoxP- SpHIS5 BcBAPAT<-, <-ScPTEF1- ScTADH1, BcBAPAT<-PTEF1- SpHIS5EcYdfG-> ScPPGK1-> ScTCYC1 PPGK1->EcYdfG pXII-1-LoxP- pXII-1-LoxP-KlLEU2 ScAAT2<- ScPTEF1<- ScTADH1 KlLEU2-AAT2<-PTEF1 KlLEU2 (SEQ ID NO88) *pTY, a vector designed for multiple chromosomal integration bytargeting TY repeat regions.

The vector contains the same USER cloning cassette as the rest of theparent plasmids listed in Table 4.

TABLE 5 Strains and 3HP titers in cultivation with β- alanine additionPlasmid with URA3 Plasmid with HIS3 Plasmid with LEU2 3HP, Parent strainmarker marker marker mg/L CEN.PK113-11C (ura- pPpBAPAT pPaHIBADH — −10 ±2  his-) CEN.PK113-11C (ura- pPpBAPAT pCaHIBADH — −16 ± 3  his-)CEN.PK113-11C (ura- pPpBAPAT pBcHIBADH — −11 ± 5  his-) CEN.PK113-11C(ura- pPpBAPAT pPpHIBADH — −10 ± 5  his-) CEN.PK113-11C (ura- pPpBAPATpMsHPDH — −12 ± 6  his-) CEN.PK113-11C (ura- pPpBAPAT pStMSR — −4 ± 4his-) CEN.PK113-11C (ura- pPpBAPAT pEcRutE — −6 ± 5 his-) CEN.PK113-11C(ura- pPpBAPAT pEcYdfG — −14 ± 2  his-) CEN.PK113-11C (ura- pBcBAPATpPaHIBADH — 474 ± 15 his-) CEN.PK113-11C (ura- pBcBAPAT pCaHIBADH — 489± 73 his-) CEN.PK113-11C (ura- pBcBAPAT pBcHIBADH — 434 ± 29 his-)CEN.PK113-11C (ura- pBcBAPAT pPpHIBADH — 496 ± 14 his-) CEN.PK113-11C(ura- pBcBAPAT pMsHPDH — 1,852 ± 103  his-) CEN.PK113-11C (ura- pBcBAPATpStMSR — 1,445 ± 40   his-) CEN.PK113-11C (ura- pBcBAPAT pEcRutE — 394 ±8  his-) CEN.PK113-11C (ura- pBcBAPAT pEcYdfG — 2,145 ± 89   his-)CEN.PK102-5B.URA3 — pPaHIBADH pCaGabT −7 ± 4 (his-leu-)CEN.PK102-5B.URA3 — pCaHIBADH pCaGabT −1 ± 5 (his-leu-)CEN.PK102-5B.URA3 — pBcHIBADH pCaGabT  19 ± 20 (his-leu-)CEN.PK102-5B.URA3 — pPpHIBADH pCaGabT −9 ± 0 (his-leu-)CEN.PK102-5B.URA3 — pMsHPDH pCaGabT −9 ± 4 (his-leu-) CEN.PK102-5B.URA3— pStMSR pCaGabT −5 ± 4 (his-leu-) CEN.PK102-5B.URA3 — pEcRutE pCaGabT 6 ± 2 (his-leu-) CEN.PK102-5B.URA3 — pEcYdfG pCaGabT −10 ± 2 (his-leu-) CEN.PK102-5B.URA3 — pPaHIBADH pScGabT 233 ± 17 (his-leu-)CEN.PK102-5B.URA3 — pCaHIBADH pScGabT 205 ± 29 (his-leu-)CEN.PK102-5B.URA3 — pBcHIBADH pScGabT 191 ± 19 (his-leu-)CEN.PK102-5B.URA3 — pPpHIBADH pScGabT 202 ± 11 (his-leu-)CEN.PK102-5B.URA3 — pMsHPDH pScGabT 493 ± 23 (his-leu-)CEN.PK102-5B.URA3 — pStMSR pScGabT 435 ± 23 (his-leu-) CEN.PK102-5B.URA3— pEcRutE pScGabT 170 ± 11 (his-leu-) CEN.PK102-5B.URA3 — pEcYdfGpScGabT 457 ± 18 (his-leu-)

TABLE 6 Strains and 3HP titers in cultivation with L- aspartate additionPlasmid with URA3 Plasmid with HIS3 3HP, Parent strain marker markermg/L CEN.PK113-11C (ura-his-) pESC-URA-BcBAPAT- −1 ± 0  MsHDPHpESC-HIS-EcPanD CEN.PK113-11C (ura-his-) pESC-URA-BcBAPAT- 79 ± 8 MsHDPH pESC-HIS-CgPanD CEN.PK113-11C (ura-his-) pESC-URA-BcBAPAT- 0 ± 0MsHDPH pESC-HIS-ScGAD1 CEN.PK113-11C (ura-his-) pESC-URA-BcBAPAT- 0 ± 0MsHDPH pESC-HIS-EcGAD1 CEN.PK113-11C (ura-his-) pESC-URA-BcBAPAT- −1 ±0  MsHDPH pESC-HIS-RnGAD1 CEN.PK113-11C (ura-his-) pESC-URA-BcBAPAT- −1± 0  EcYdfG pESC-HIS-EcPanD CEN.PK113-11C (ura-his-) pESC-URA-BcBAPAT-269 ± 53  EcYdfG pESC-HIS-CgPanD CEN.PK113-11C (ura-his-)pESC-URA-BcBAPAT- 0 ± 0 EcYdfG pESC-HIS-ScGAD1 CEN.PK113-11C (ura-his-)pESC-URA-BcBAPAT- 0 ± 0 EcYdfG pESC-HIS-EcGAD1 CEN.PK113-11C (ura-his-)pESC-URA-BcBAPAT- 0 ± 1 EcYdfG pESC-HIS-RnGAD1 CEN.PK113-5D (ura-)pESC-URA-BcBAPAT- — 1 ± 0 EcYdfG CEN.PK113-7D (WT) — — 0 ± 0

TABLE 7 Strains and 3HP titers in cultivation on glucose as the solecarbon source Plasmid with URA3 Plasmid with HIS3 3HP, mg/L on 3HP, mg/Lon Parent strain marker marker Delft medium FIT medium CEN.PK113-11C(ura- pESC-URA-BcBAPAT- pESC-HIS-TcPanD 605 ± 18 1638 ± 19 his-) EcYdfGCEN.PK113-11C (ura- pESC-URA-BcBAPAT- pESC-HIS-CgPanD 214 ± 32  826 ± 33his-) EcYdfG

TABLE 8 Yeast strains with chromosomally integrated genes for 3HPbiosynthesis Plasmid with URA3 Plasmid with Plasmid with Final strainParent strain marker HIS3 marker LEU2 marker SCE-R2-180 CEN.PK102-5B(ura- pX-2-LoxP-KlURA3- pX-4-LoxP- pXII-1-LoxP- his-leu-)BcBAPAT<-PTEF1- SpHiS5-TcPanD<- KlLEU2-AAT2<- PPGK1->EcYdfG PTEF1 PTEF1SCE-R2-182 CEN.PK102-5B (ura- pTY-BcBAPATC-PTEF1- pX-4-LoxP-pXII-1-LoxP- his-leu-) PPGK1->EcYdfG SpHiS5-TcPanD<- KlLEU2-AAT2<- PTEF1PTEF1 SCE-R2-184 pTY-TcPanD<-PTEF1 pX-4-LoxP- pXII-1-LoxP- CEN.PK102-5B(ura- SpHiS5-BcBAPAT<- KlLEU2-AAT2<- his-leu-) PTEF1-PPGK1-PTEF1 >EcYdfG SCE-R2-188 CEN.PK113-11C (ura- pX-2-LoxP-KlURA3-pX-4-LoxP- — his-) BcBAPATC-PTEF1- SpHiS5-TcPanD<- PPGK1->EcYdfG PTEF1SCE-R2-190 CEN.PK113-11C (ura- pTY-BcBAPATC-PTEF1- pX-4-LoxP- — his-)PPGK1->EcYdfG SpHiS5-TcPanD<- PTEF1 SCE-R2-192 CEN.PK113-11C (ura-pTY-TcPanD<-PTEF1 pX-4-LoxP- — his-) SpHiS5-BcBAPAT<-PTEF1-PPGK1- >EcYdfG SCE-R2-196 ST738 pX-2-LoxP-KlURA3- pX-4-LoxP-pXII-1-LoxP- (PYC1{circumflex over ( )}, PYC2{circumflex over ( )}, ura-BcBAPAT<-PTEF1- SpHiS5-TcPanD<- KlLEU2-AAT2<- his-leu-) PPGK1->EcYdfGPTEF1 PTEF1 SCE-R2-198 ST738 pTY-BcBAPATC-PTEF1- pX-4-LoxP- pXII-1-LoxP-(PYC1{circumflex over ( )}, PYC2{circumflex over ( )}, ura-PPGK1->EcYdfG SpHiS5-TcPanD<- KlLEU2-AAT2<- his-leu-) PTEF1 PTEF1SCE-R2-200 ST738 pTY-TcPanD<-PTEF1 pX-4-LoxP- pXII-1-LoxP-(PYC1{circumflex over ( )}, PYC2{circumflex over ( )}, ura-SpHiS5-BcBAPAT<- KlLEU2-AAT2<- his-leu-) PTEF1-PPGK1- PTEF1 >EcYdfGSCE-R2-204 ST724 pX-2-LoxP-KlURA3- pX-4-LoxP- — (PYC1{circumflex over( )}, PYC2{circumflex over ( )}, ura- BcBAPAT<-PTEF1- SpHiS5-TcPanD<-his-) PPGK1->EcYdfG PTEF1 SCE-R2-206 ST724 pTY-BcBAPATC-PTEF1-pX-4-LoxP- — (PYC1{circumflex over ( )}, PYC2{circumflex over ( )}, ura-PPGK1->EcYdfG SpHiS5-TcPanD<- his-) PTEF1 SCE-R2-208 ST724pTY-TcPanD<-PTEF1 pX-4-LoxP- — (PYC1{circumflex over ( )},PYC2{circumflex over ( )}, ura- SpHiS5-BcBAPAT<- his-) PTEF1-PPGK1->EcYdfG

Results obtained in the following Examples are in part given in theaccompanying drawings, in which:

FIG. 1 shows a metabolic pathway leading from pyruvate to 3-HP viaaspartate and beta-alanine and malonic semialdehyde.

FIG. 2 shows NMR results obtained in Example 2.

FIG. 3 shows the influence of integrating multiple copies of genes andof overexpression of precursor supply genes on 3HP titer. Theconcentration of 3HP in the culture broth was determined by HPLC methodand is given in g L⁻¹. ↑-single copy of gene is integrated into thegenome, ↑↑-multiple copies of gene are integrated into the genome(Example 6).

FIG. 4 shows growth and metabolite concentrations in glucose-limitedfed-batch cultivation of SCE-R2-200 at pH5. Representative graph of onecultivation out of three (Example 7).

As illustrated in FIG. 1, apartate can be converted to beta-alanine bythe enzyme PanD, aspartate 1-decarboxylase. β-alanine is convertible tomalonic semialdehyde by either BAPAT or GabT, and malonic semialdehydeis convertible to 3-HP by HIBADH/HPDH. The present invention uses theroute via BAPAT.

EXAMPLE 1 Cloning of heterologous beta-alanine-pyruvateaminotransferase, 3-hydroxyisobutyrate dehydrogenase, and3-hydroxypropanoate Dehydrogenase and Overexpession of Heterologous andNative Gamma-Aminobutyrate Transaminase in S. cerevisiae

Genes encoding a putative B. cereus aminotransferase yhxA (SEQ ID NO1),Pseudomonas putida beta-alanine-pyruvate aminotransferase (SEQ ID NO3),P. aeruginosa 3-hydroxybutyrate dehydrogenase (SEQ ID NO5), Candidaalbicans 3-hydroxybutyrate dehydrogenase (SEQ ID NO7), P. putida3-hydroxybutyrate dehydrogenase (SEQ ID NO9), Bacillus cereus3-hydroxybutyrate dehydrogenase (SEQ ID NO11), Metallosphaera sedula3-hydroxypropanoate dehydrogenase (SEQ ID NO13), Sulfolobus tokadaii3-hydroxypropanoate dehydrogenase (SEQ ID NO15), and Clostridiumacetobutylicum gamma-aminobutyrate transaminase (SEQ ID NO17) weresynthesized by GeneArt (Life Technologies) in versions codon-optimizedfor yeast S. cerevisiae (corresponding SEQ ID NO2, SEQ ID NO4, SEQ IDNO6, SEQ ID NO8, SEQ ID NO10, SEQ ID NO12, SEQ ID NO14, SEQ ID NO16, SEQID NO18).

The ordered gene constructs had a general structure: GGTACCAAAACAATGNN .. . NNTGAGTCGAC (SEQ ID NO67), where GGTACC is a KpnI restriction site,AAAACA is the Kozak sequence, ATG is the start codon, NN . . . NNrepresents the protein coding sequence without start and stop codons,TGA is the stop codon, GTCGAC is a SalI restriction site.

The synthetic genes were excised from the plasmids using KpnI and SalI,gel-purified and ligated into plasmid pE1 (SEQ ID 81) or pE2 (SEQ ID82),which were digested with the same enzyme pair. The resulting ligationmix was transformed into chemically competent E. coli DH5alpha usingheat shock and the cells were selected on Luria-Bertani (LB) agar mediumwith 100 μg/ml amplicillin.

The clones with correct inserts were identified by colony PCR,inoculated in liquid LB medium with 100 μg/ml ampicillin and theplasmids were isolated (Table 2). The resulting plasmids were confirmedby sequencing.

The gene fragments carrying the genes and correct overhangs forUSER-cloning were generated by PCR amplification using primers andtemplates as indicated in Table 3. The PCR mix contained: 28 μl water,10 μl high fidelity Phusion® polymerase buffer (5×), 5 μl 2 mM dNTP, 1μl Phusion® polymerase, 2.5 μl forward primer at 10 μM concentration,2.5 μl reverse primer at 10 μM concentration, and 1 μl DNA template. Thecycling program was: 95° C. for 2 min, 30 cycles of [95° C. for 10 sec,50° C. for 20 sec, 68° C. for 2 min], 68° C. for 5 min, pause at 10° C.The gene fragments were resolved on 1% agarose gel containing SYBR®-SAFE(Invitrogen) and purified using NucleoSpin® Gel and PCR Clean-up kit(Macherey-Nagel). The promoter fragments were also generated by PCRfollowed by gene purification (Table 3). The terminators were alreadypresent on the expression plasmids.

The parent plasmids pESC-Ura-USER (SEQ ID NO 85), pESC-His-USER (SEQ IDNO 83) and pESC-Leu-USER (SEQ ID NO 84) were linearized with FastDigest®AsiSI (Fermentas) for 1 hour at 37° C. and nicked with Nb.BsmI for 1hour at 37° C. The resulting linearized nicked DNA was purified from thesolution and eluted in 5 mM Tris buffer, pH 8.0.

The expression plasmids were created by USER-cloning using the followingprotocol. 1 μl of linearized and nicked parent plasmid was mixed with 1μl of promoter fragment, 2 μl of gene fragment, 0.5 μl Taq polymerasebuffer, 0.5 μl USER enzyme (NEB). The mix was incubated at 37° C. for 25min, at 25° C. for 25 min and transformed into chemically competent E.coli DH5alpha. The clones with correct inserts were identified by colonyPCR and the plasmids were isolated from overnight E. coli cultures andconfirmed by sequencing. The expression plasmids are listed in Table 4.

The expression plasmids were transformed into S. cerevisiae cells usingthe lithium acetate transformation protocol. The cells were selected onsynthetic complete (SC) agar medium without uracil, histidine andleucine. The resulting strains are listed in Table 5.

EXAMPLE 2 Production of 3-hydroxypropionic Acid in S. cerevisiaeCultivated on β-Alanine

At least four independent yeast transformants were streak-purified on SCura-his-leu-agar plates. Four single colonies originating fromindependent transformants were inoculated in 0.5 ml SC ura-his-leu- in a96-deep well microtiter plate with air-penetrable lid (EnzyScreen). Theplates were incubated at 30° C. with 250 rpm agitation at 5 cm orbitcast overnight. 50 μl of the overnight cultures were used to inoculate0.5 ml minimal mineral (Delft) medium with 10 g/L β-alanine in a 96-deepwell plate.

The composition of the of Delft medium was as following: 7.5 g(NH₄)₂SO₄, 14.4 g KH₂PO₄, 0.5 g MgSO₄.7H₂O, 22 g dextrose, 2 mL tracemetals solution, and 1 mL vitamins. pH of the medium was adjusted to 6.The trace metals solution contained per liter: 4.5 g CaCl₂.2H₂O, 4.5 gZnSO₄.7H₂O, 3 g FeSO₄.7H₂O, 1 g H₃BO₃, 1 g MnCl₂.4H₂O, 0.4 gNa₂MoO₄.2H₂O, 0.3 g CoCl₂.6H₂O, 0.1 g CuSO₄.5H₂O, 0.1 g KI, 15 g EDTA.The trace metals solution was prepared by dissolving all the componentsexcept EDTA in 900 mL ultra-pure water at pH 6 followed by gentleheating and addition of EDTA. Finally the trace metal solution pH wasadjusted to 4, and the solution volume was adjusted to 1 L andautoclaved (121° C. in 20 min). Trace metals solution was stored at +4°C. The vitamins solution contained per liter: 50 mg biotin, 200 mgp-aminobenzoic acid, 1 g nicotinic acid, 1 g Ca-pantotenate, 1 gpyridoxine-HCl, 1 g thiamine-HCl, 25 g myo-inositol. Biotin wasdissolved in 20 mL 0.1 M NaOH and 900 mL water is added. pH was adjustedto 6.5 with HCl and the rest of the vitamins was added. pH wasre-adjusted to 6.5 just before and after adding m-inositol. The finalvolume of the vitamin solution was adjusted to 1 l and sterile-filteredbefore storage at +4° C.

Fermentation was carried out for 72 hours at the same conditions asabove.

At the end of the cultivation the OD₆₀₀ was measured. 10 μl of thesample was mixed with 190 μl water and absorbance was measured at 600 nmwave length in spectrophotometer (BioTek).

The culture broth was spun down and the supernatant analyzed for3-hydroxypropionic acid concentration using enzymatic assay (Table 5).No 3HP production was obtained when P. putida beta-alanine-pyruvateaminotransferase or C. acetobutylicum gamma-aminobutyrate transaminasewere used in combination with 3-hydroxybutyrate dehydrogenase or3-hydroxypropanoate dehydrogenase. However 3HP production frombeta-alanine was observed when putative B. cereus aminotransferase YhxAor S. cerevisiae gamma-aminobutyrate transaminase were combined with3-hydroxybutyrate dehydrogenase or 3-hydroxypropanoate dehydrogenase(Table 5: strains 133-147). The best enzyme combination under theconditions tested was strain 147 expressing B. cereus aminotransferaseYhxA and E. coli 3-hydroxypropanoate dehydrogenase YdfG, where 2,145±89mg/L 3HP was obtained.

Enzymatic assay was carried out as follows. 20 μl of standards (3HP atconcentrations from 0.03 to 1 g/L in Delft medium) and samples wereadded to a 96-well flat bottom transparent plate (Greiner). 180 μl ofmix (14.8 ml water, 2 ml buffer (1 mM Tris, 25 mM MgCl₂, pH 8.8), 1 mlNADP+ solution (50 mg/ml), and 0.2 ml purified YdfG enzyme in PBS buffer(1500 μg/ml)) was added per well using multichannel pipet. The startabsorbance at 340 nm was measured, the plate was sealed and incubated at30° C. for 1.5 hours. After that the end absorbance at 340 nm wasmeasured again. The difference between the end and the start valuescorrected for the background were in linear correlation with 3HPconcentrations. The concentration of 3HP in the samples was calculatedfrom the standard curve.

The identity of 3-hydroxypropionic acid in the best sample was confirmedby NMR analysis (FIG. 2). The concentration measured by NMR correlatedwell with the value found by enzymatic assay.

EXAMPLE 3 Cloning of aspartate-1-decarboxylase or GlutamateDecarboxylase in S. cerevisiae

Genes encoding E. coli aspartate 1-decarboxylase (SEQ ID NO50) and C.glutamicum aspartate 1-decarboxylase (SEQ ID NO52) were synthesized asgBLOCKs by Integrated DNA Technologies (in versions codon-optimized foryeast S. cerevisiae corresponding SEQ ID NO51 and SEQ ID NO53).

Gene encoding glutamate decarboxylase from Rattus norvegicus (SEQ IDNO58) was synthesized by GeneArt (Life Technologies) in versioncodon-optimized for yeast S. cerevisiae (SEQ ID NO59).

The ordered gene constructs had a general structure: GGTACCAAAACAATGNN .. . NNTGAGTCGAC (SEQ ID NO67), where GGTACC is a KpnI restriction site,AAAACA is the Kozak sequence, ATG is the start codon, NN . . . NNrepresents the protein coding sequence without start and stop codons,TGA is the stop codon, GTCGAC is a SalI restriction site.

The gene fragments carrying the genes and correct overhangs forUSER-cloning were generated by PCR amplification using primers andtemplates as indicated in Table 3. The PCR mix contained: 28 μl water,10 μl high fidelity Phusion® polymerase buffer (5×), 5 μl 2 mM dNTP, 1μl Phusion® polymerase, 2.5 μl forward primer at 10 μM concentration,2.5 μl reverse primer at 10 μM concentration, and 1 μl DNA template. Thecycling program was: 95° C. for 2 min, 30 cycles of [95° C. for 10 sec,50° C. for 20 sec, 68° C. for 2 min], 68° C. for 5 min, pause at 10° C.The gene fragments were resolved on 1% agarose gel containing SYBR®-SAFE(Invitrogen) and purified using NucleoSpin® Gel and PCR Clean-up kit(Macherey-Nagel). The promoter fragments were also generated by PCRfollowed by gene purification (Table 3). The terminators were alreadypresent on the expression plasmids.

The parent plasmids pESC-Ura-USER, pESC-His-USER and pESC-Leu-USER werelinearized with FastDigest® AsiSI (Fermentas) for 1 hour at 37° C. andnicked with Nb.BsmI for 1 hour at 37° C. The resulting linearized nickedDNA was purified from the solution and eluted in 5 mM Tris buffer, pH8.0.

The expression plasmids were created by USER-cloning using the followingprotocol. 1 μl of linearized and nicked parent plasmid was mixed with 1μl of promoter fragment, 2 μl of gene fragment, 0.5 μl Taq polymerasebuffer, 0.5 μl USER enzyme (NEB). The mix was incubated at 37° C. for 25min, at 25° C. for 25 min and transformed into chemically competent E.coli DH5alpha. The clones with correct inserts were identified by colonyPCR and the plasmids were isolated from overnight E. coli cultures andconfirmed by sequencing. The expression plasmids are listed in Table 4.

The expression plasmids were transformed into S. cerevisiae cells usingthe lithium acetate transformation protocol. The cells were selected onsynthetic complete (SC) agar medium without uracil, histidine andleucine. The resulting strains are listed in Table 6.

EXAMPLE 4 Production of 3-hydroxypropionate in S. cerevisiae Cultivatedon L-Aspartate

At least four independent yeast transformants were streak-purified on SCura-his-leu-agar plates. Four single colonies originating fromindependent transformants were inoculated in 0.5 ml SC ura-his-leu- in a96-deep well microtiter plate with air-penetrable lid (EnzyScreen). Theplates were incubated at 30° C. with 250 rpm agitation at 5 cm orbitcast overnight. 50 μl of the overnight cultures were used to inoculate0.5 ml Delft medium with 10 g/L L-aspartate in a 96-deep well plate.Fermentation was carried out for 72 hours at the same conditions asabove.

The culture broth was spun down and the supernatant analyzed for3-hydroxypropionic acid concentration using enzymatic assay as describedin Example 2 (Table 6).

3HP production from L-aspartate was observed only when aspartate1-decarboxylase from C. glutamicum was expressed in combination withenzymes converting beta-alanine into 3HP (putative B. cereusaminoransferase YhxA and E. coli 3-hydroxypropanoate dehydrogenase YdfGor Metallosphaera sedula 3-hydroxypropanoate dehydrogenase). The bestcombination was aspartate 1-decarboxylase from C. glutamicum, putativeB. cereus aminoransferase YhxA and E. coli 3-hydroxypropanoatedehydrogenase YdfG, which resulted in 269±53 mg/L 3HP.

In this specification, unless expressly otherwise indicated, the word‘or’ is used in the sense of an operator that returns a true value wheneither or both of the stated conditions is met, as opposed to theoperator ‘exclusive or’ which requires that only one of the conditionsis met. The word ‘comprising’ is used in the sense of ‘including’ ratherthan in to mean ‘consisting of’. All prior teachings acknowledged aboveare hereby incorporated by reference. No acknowledgement of any priorpublished document herein should be taken to be an admission orrepresentation that the teaching thereof was common general knowledge inAustralia or elsewhere at the date hereof.

EXAMPLE 5 Expression of aspartate-1-decarboxylase from red Flour Beetlein S. cerevisiae and Production of 3HP from Glucose

The gene encoding Tribolium castaneum aspartate 1-decarboxylase TcPanD(SEQ ID 68) was synthesized in version codon-optimized for S. cerevisiae(SEQ ID 69) by GeneArt (LifeTech Sciences).

The TcPanD gene was amplified using PCR in order to generateUSER-cloning compatible overhangs as described in Example 1 usingprimers TcPanD_U1_fw and Tc_PanD_rv (Table 3). The resulting DNAfragment TcPanD<—was cloned into expression plasmid pESC-HIS-USER alongwith TEF1 promoter to result in plasmid pESC-HIS-TcPanD (Table 4).Correct insertion of TcPanD gene and the promoter was confirmed bysequencing.

The plasmids were transformed into S. cerevisiae strain using thelithium acetate transformation protocol; the resulting strains are shownin Table 7.

At least three independent yeast transformants were inoculated in 0.5 mlSC ura-his-leu- in a 96-deep well microtiter plate with air-penetrablelid (EnzyScreen). The plates were incubated at 30° C. with 250 rpmagitation at 5 cm orbit cast overnight. 50 μl of the overnight cultureswere used to inoculate 0.5 ml minimal mineral (Delft) medium or 0.5 mlFeed-in-time medium (FIT) for S. cerevisiae (M2P Labs, Germany) in96-deep well plates.

Fermentation was carried out for 72 hours at the same conditions asinoculum preparation. The culture broth was spun down and thesupernatant was analyzed for 3-hydroxypropionic acid concentration usingHPLC (Table 7).

HPLC analysis was performed on Dionex UltiMate 3000 system (ThermoFisher Scientific) with Aminex HPX-87H column (Bio-Rad Laboratories,Hercules, Calif.) operating at 60° C. The injection volume was 20 μl.The mobile phase was 1 mM H₂SO₄ at a flow rate of 0.6 ml/min. 3HP wasdetected on DAD-3000 Diode Array Detector (Dionex) using the read at 210nm. The calibration curve was made using 3-hydroxypropionic acidpurchased from TCI. The identity of the 3-hydroxypropionic acid wasadditionally verified by comparison of the spectrum with the standard.

Aspartate 1-decarboxylase from T. castaneum resulted in almost 3-foldhigher 3HP titer on Delft and 2-fold higher 3HP titer on FIT medium thanaspartate 1-decarboxylase from C. glutamicum. Thus we have confirmedthat if the strain capable of producing 3HP from β-alanine issupplemented with aspartate 1-decarboxylase enzyme from C. glutamicum orbetter from T. castaneum then it can produce 3HP directly from glucose.

EXAMPLE 6 Improvement of 3HP Production by Overexpression of Precursor

Once the biosynthesis of 3HP from glucose via beta-alanine has beenestablished in yeast, the next goal was to improve the expression of thebiosynthetic genes and to increase the flux towards L-aspartate. As thiswould require stable simultaneous overexpression of several genes, weused EasyClone integrative vectors for yeast. We tested the effect ofoverexpressing native cytoplasmic aspartate aminotransferase Aat2p,pyruvate carboxylases Pyc1p and Pyc2p and of the combination thereof. Wealso investigated the effect of multiple chromosomal integration of thekey biosynthetic genes leading from aspartate to 3HP.

The genes encoding aspartate aminotransferase AAT2 and pyruvatecarboxylases PYC1 and PYC2 were amplified from gDNA of S. cerevisiaeCEN.PK113-7D using primers as in Table 3 and PCR conditions as inExample 1. The resulting DNA fragments were purified and cloned intoEasyClone expression vectors as described in Example 1 (see Table 4).

Strain ST724 (PYC1̂, PYC2̂, ura-his-) was created by transforming S.cerevisiae CEN.PK102-5B (ura-his-leu-) with plasmidpXI-1-LoxP-KlLEU2-PYC1<-PTEF1-PPGK1->PYC2, selecting the transformantson SC drop-out medium without leucine and confirming the correctintegration of the plasmid by PCR on genomic DNA of the transformant.Strain ST724 was used to create strain ST738 (PYC1̂, PYC2̂, ura-his-leu-)by looping out the KlLEU2 selection marker using LoxP-Cre-mediatedrecombination.

The yeast strains were transformed with expression plasmids according toTable 8 and transformants were selected on SC drop-out medium withouturacil, histidine and leucine. The strains were cultivated and 3HPconcentrations were analyzed as described in Example 5. The results areshown in FIG. 3.

Increasing copy number of BcBAPAT/EcYdfG or of TcPanD lead toimprovement of 3HP titer for all the four background strains tested(reference, overexpressing AAT2, overexpressing PYC1&PYC2 andoverexpressing AAT2&PYC1&PYC2). The effect of multiple integrations ofTcPanD was larger than that of multiple copies of BcBAPAT/EcYdfG.

The increased precursor supply (via overexpression of PYC1/PYC2 and/orAAT2) had a positive effect on 3HP production in strains with multiplecopies of TcPanD or BcBAPAT/EcYdfG genes, but not in the strains thathad only single copies of the latter genes. The positive effect ofoverexpressing pyruvate carboxylase genes was only observed onfeed-in-time medium, which simulates fed-batch conditions. The highesttiters were obtained for the strain SCE-R2-200(AAT2↑PYC1↑PYC2↑BcBAPAT↑EcYdfG↑TcPanD↑↑): 1.27±0.28 g/L and 8.51±1.05g/L on mineral and feed-in-time media correspondingly.

EXAMPLE 7 Production of 3HP by Yeast in Fed-Batch Cultivation at pH5

The best isolate of strain SCE-R2-200 described above was cultivated inaerobic fed-batch cultivation with glucose-limited feed at pH5 intriplicates.

SCE-R2-200 glycerol stock (0.3 ml) was inoculated in 150 ml Delft mediumin 500-ml baffled shake flask and propagated at 30° C. with 250 rpmagitation for about 24 hours. The culture was concentrated down to 50 mlby centrifugation at 4,000×g for 2 min and used to inoculate 0.5 Lmedium in 1L-Sartorius reactor. The final medium in the reactorscontained per liter: 15 g (NH₄)₂SO₄, 6 g KH₂PO₄, 1 g MgSO₄.7H₂O, 4 mltrace metals solution, 2 ml vitamins solution, 0.4 ml antifoam A(Sigma-Aldrich), and 44 g dextrose. Dextrose was autoclaved separately,vitamins solution was sterile filtered and added to the medium afterautoclavation. The trace metal and vitamins solutions are the same asdescribed in Example 2. The agitation rate was 800 rpm, the temperaturewas 30° C., aeration was 1 L min⁻¹ air and pH was maintained at 5.0 byautomatic addition of 2N NaOH. Carbon dioxide concentration in theoff-gas was monitored by acoustic gas analyzer (model number 1311, Bruël& Kjær). Once the glucose was exhausted, which was observed from declinein CO₂ production and was also confirmed by residual glucose detectionusing glucose strips Glucose MQuant™ (Merck Millipore), the feed wasstarted at 5 g h⁻¹. The feed contained per liter: 45 g (NH₄)₂SO₄, 18 gKH₂PO₄, 3 g MgSO₄.7H₂O, 12 ml trace metals solution, 6 ml vitaminssolution, 0.6 ml antifoam A, and 176 g dextrose. Dextrose was autoclavedseparately, vitamins solution was sterile filtered and added to the feedafter autoclavation.

24 hours after the feed start the feed rate was ramped up to 10 g h⁻¹and 48 hours after the feed start it was further increased to 15 g h⁻¹.The reactors were sampled twice a day to measure biomass dry weight andmetabolites. For metabolites analysis the sample was immediatelycentrifuged and the supernatant was stored at −20° C. until HPLCanalysis. HPLC analysis of glucose, succinate, acetate, 3HP, glycerol,ethanol, and pyruvate was carried out at described in Example 5.Glucose, glycerol and ethanol were detected using RI-101 RefractiveIndex Detector (Dionex). 3HP, pyruvate, succinate and acetate weredetected with DAD-3000 Diode Array Detector at 210 nm (Dionex).

The strain produced 3-hydroxypropionic acid at 13.7±0.3 g·L-1 titer,14±0% C-mol·C-mol-1 glucose yield and 0.24±0.0 g·L-1·h-1 productivity.No significant amounts of by-products as acetate, ethanol or glycerolwere detected at the end of the fermentation. Results are shown in FIG.4.

In this specification, unless expressly otherwise indicated, the word‘or’ is used in the sense of an operator that returns a true value wheneither or both of the stated conditions is met, as opposed to theoperator ‘exclusive or’ which requires that only one of the conditionsis met. The word ‘comprising’ is used in the sense of ‘including’ ratherthan in to mean ‘consisting of’. All prior teachings acknowledged aboveare hereby incorporated by reference. No acknowledgement of any priorpublished document herein should be taken to be an admission orrepresentation that the teaching thereof was common general knowledge inAustralia or elsewhere at the date hereof. The content of the sequencelisting filed herewith forms part of the description of the invention.

1. A genetically modified yeast cell comprising an active fermentationpathway producing 3-HP, wherein the cell comprises and expresses anexogenous gene coding for the production of an enzyme having at least80% identity with SEQ ID NO: 1 and catalysing a transamination reactionbetween beta-alanine and pyruvate to produce malonate semialdehyde.
 2. Agenetically modified yeast cell as claimed in claim 1, wherein saidenzyme is the aminotransferase YhxA from Bacillus cereus AH1272.
 3. Agenetically modified yeast cell as claimed in claim 1, expressing a3-hydroxyisobutyrate dehydrogenase (HIBADH).
 4. A genetically modifiedyeast cell as claimed in claim 3, wherein said HIBADH is fromPseudomonas aeruginosa, P. putida, Bacillus cereus, or Candida albicans.5. A genetically modified yeast cell as claimed in claim 1, expressing a3-hydroxypropanoate dehydrogenase (3-HPDH).
 6. A genetically modifiedyeast cell as claimed in claim 5, wherein said 3-HPDH is fromMetallosphaera sedula, Sulfolobus tokadaii or E. coli.
 7. A geneticallymodified yeast cell as claimed in claim 1 expressing an exogenous geneproducing an aspartate-1-decarboxylase (EC 4.1.1.11) and/or expressingan exogenous gene expressing a glutamate decarboxylase (EC 4.1.1.15). 8.A genetically modified yeast cell as claimed in claim 7 expressingaspartate-1-decarboxylase from Corynebacterium glutamicum (SEQ ID NO52)or an enzyme having aspartate-1-decarboxylase activity which is at least80% homologous with SEQ ID NO52.
 9. A genetically modified yeast cell asclaimed in claim 7 expressing aspartate-1-decarboxylase from Triboliumcastaneum (SEQ ID NO68) or an enzyme having aspartate-1-decarboxylaseactivity which is at least 80% homologous with SEQ ID NO68.
 10. Agenetically modified yeast cell as claimed in claim 7 with increasedactivity of pyruvate carboxylase and/or aspartate transaminase.
 11. Agenetically modified yeast cell as claimed in claim 10 overexpressingnative pyruvate carboxylase PYC1 or PYC2 and/or native aspartateaminotransferase AAT2.
 12. A genetically modified yeast cell as claimedin claim 1, wherein the yeast is S. cerevisiae.
 13. A method for theproduction of 3HP comprising culturing a genetically modified yeast cellcomprising an active fermentation pathway producing 3-HP, wherein thecell comprises and expresses an exogenous gene coding for the productionof an enzyme having at least 80% identity with SEQ ID NO: 1 andcatalysing a transamination reaction between beta-alanine and pyruvateto produce malonate semialdehyde, and recovering 3HP from the culture.14. A method as claimed in claim 13, comprising supplying said culturewith beta-alanine and/or L-aspartate.
 15. A method as claimed in claim13, wherein at least 100 mg of 3HP per litre of culture medium isproduced or is recovered from said culture medium.